Methods are already known for the fluorescence spectroscopic determination of a biological substance provided with a marker consisting of a lanthanide chelate complex, formed of a lanthanide metal ion such as a europium ion or terbium ion coupled to the substance via a chelate-forming compound such as ethylene diamine tetraacetic acid (EDTA) or an analogue thereof, by excitation by a short radiation pulse and detection of the fluorescence of the marker when the fluorescence from any noise source has substantially ceased. These methods have application in time-resolved fluorometric assays using labelled tracer molecules such as labelled antigens or antibodies or nucleic acid probes.
U.S. Pat. No. 4,565,790 and European Patent 0,064,484 disclose an improvement in such a process in which, before the determination is carried out, a solution of low pH, which suitably brings the pH to 3.5 or below and contains a detergent and a .beta.-diketone, is added to dissociate the lanthanide ion from the chelate complex and to transfer the dissociated lanthanide ion into a fluorescent form, whereupon the determination is performed in the solution.
As described in those patents the solution added to reduce the pH necessarily contains a detergent and the determination of the fluorescence is necessarily carried out at the low pH, said to be suitably 3.5 or less. Also, the only disclosure of the addition of .beta.-diketone is as part of the low pH solution. The present invention seeks, inter alia, to improve that process.